Structure and biology of complement protein C3, a connecting link between innate and acquired immunity

Arvind Sahu Structure and biology ofJohn D. Lambris

complement protein C3, aconnecting link between innate andacquired immunity

Authors’ addresses

Arvind Sahu1, John D. Lambris2,1National Centre for Cell Science, PuneUniversity Campus, Ganeshkhind, Pune, India.2Protein Chemistry Laboratory, Department ofPathology and Laboratory Medicine,University of Pennsylvania, Philadelphia,Pennsylvania, USA.

Correspondence to:

John D. LambrisProtein Chemistry LaboratoryDepartment of Pathology and LaboratoryMedicine401 Stellar-Chance LaboratoriesUniversity of PennsylvaniaPhiladelphia PA 19104USAFax: 1 215 573 8738e-mail: [email protected]

Acknowledgements

A.S. is a Wellcome Trust Overseas SeniorResearch Fellow in Biomedical Science inIndia. This research was supported byNational Institutes of Health grants AI 30040,GM 56698, HL28220, and AI 48487.

Immunological Reviews 2001Vol. 180: 35–48Printed in Denmark. All rights reserved

Copyright C Munksgaard 2001

Immunological ReviewsISSN 0105-2896

35

Summary: Complement protein C3 is a central molecule in the comple-ment system whose activation is essential for all the important functionsperformed by this system. After four decades of research it is now wellestablished that C3 functions like a double-edged sword: on the one handit promotes phagocytosis, supports local inflammatory responses againstpathogens, and instructs the adaptive immune response to select the ap-propriate antigens for a humoral response; on the other hand its unregu-lated activation leads to host cell damage. In addition, its interactionswith the proteins of foreign pathogens may provide a mechanism bywhich these microorganisms evade complement attack. Therefore, a clearknowledge of the molecule and its interactions at the molecular level notonly may allow the rational design of molecular adjuvants but may alsolead to the development of complement inhibitors and new therapeuticagents against infectious diseases.

Introduction

The complement system is an integral participant in the in-

nate mechanisms of immunity, in which the third component

of complement, C3, plays a central role. C3 supports the acti-

vation of all the three pathways of complement activation i.e.

the classical, alternative, and lectin pathways. Among the

complement proteins, it is probably the most versatile and

multifunctional molecule identified to date, having evolved

structural features that allow it to interact in a specific manner

with at least 25 different proteins (1). C3 is an ancient mol-

ecule; its identification in echinoderms (2) and tunicates (3)

suggests that it emerged at least 700 million years ago, long

before the appearance of immunoglobulins.

Studies of C3 began during the early part of the 20th cen-

tury. By that time, it was accepted that complement activation

proceeds in a sequential manner and that cellular intermedi-

ate products are formed during immune hemolysis. C3 activ-

ity was then considered to be due to a single component.

However, in 1958, Rapp (4) demonstrated through modified

fractionation methods that the hemolytic reaction of C3 must

involve more than one component. After the advent of chro-

Sahu & Lambris ¡ Structure and biology of C3

Table 1. Properties of human C3

Serum concentration (mg/ml) 1.2

Molecular weight

Apparenta 185,000

Calculatedb 184,342

Electrophorectic mobility b-2

Subunits (mol. wta) a chain (110,000)

b chain (75,000)

Amino acids

Signal peptide 22

Mature protein 1,637

Carbohydrate (∂%w/w) 1.5

Isoelectric point 5.7

Extinction coefficient (280 nm, 1%, 1 cm) 9.7

S20, w (Svedberg units) ∂8.3

Diffusion coefficient (10ª7 cm2/s) 4.0

Thioester hydrolysis rate (%/min) 0.005

Genetic locus 19p13.3

a Determined by SDS-PAGE.b Based on the primary sequence of the plasma protein.

matographic techniques it was discovered that C3 activity, as

defined by old assays, was actually a mixture of six individual

proteins (C3, C5, C6, C7, C8, and C9).

In the latter part of the 20th century, C3 was primarily

studied in the context of its function within the complement

system. It was shown to interact with other complement pro-

teins and with cell-surface receptors and to serve as a modu-

lator of complement-dependent leukocyte functions. Its role

in mounting an effective antibody response, though reported

(5), was overlooked. The recent availability of C3 knockout

mice has allowed us to revisit this potential role for C3. The

data obtained from studies of these mice have provided strong

supporting evidence for the paradigm that C3 forms a con-

necting link between the complement system and the ac-

quired immune response that helps trigger and modulate the

acquired immune response.

Although the interaction of C3 with other complement pro-

teins and receptors is important for the host defense, the inter-

actions of this complement component with proteins from for-

eign pathogens provide a mechanism by which these micro-

organisms can evade complement neutralization. In addition,

unregulated activation of C3, and in turn the complement sys-

tem, leads to host cell damage. Thus, elucidation of the molecu-

lar features of C3 that are important for C3–ligand interactions

is of great importance. We have devoted considerable research

effort to defining the regions of the C3 protein that are ex-

pressed at the various stages of its proteolytically controlled life

36 Immunological Reviews 180/2001

cycle and that determine the various functional roles of the C3

molecule. In this review we describe our work on the C3–

ligand interactions, C3 evolution and diversity, C3-related viral

molecular mimicry, and identification of C3-based comple-

ment inhibitors. We have also included a section on basic C3

structure and biochemistry.

C3 structure, activation, and regulation

Human C3, the most abundant complement protein in serum

(1.2 mg/ml), is comprised of a and b chains (Mr 110,000

and 75,000, respectively) (Table 1) that are connected cova-

lently by a single disulfide bond and associated by non-cova-

lent forces (Fig. 1). The protein is encoded by a 41 kb gene

located on chromosome 19 (6). The C3 gene is composed of

41 exons (ranging in size from 52–213 bases), of which 16

encode the b chain and 25 encode the a chain. The primary

structure of C3 as deduced from the cDNA sequence (7) con-

sists of 1,663 amino acids, including a 22-amino acid signal

peptide. C3 is synthesized as a single-chain pre-pro-molecule

with a and b chains linked by a tetra-arginine sequence,

which is removed by a furin-like enzyme (8) during post-

translational modification; the order of the subunits is

b–a. Following translocation through the endoplasmic

reticulum to the Golgi, N-linked high-mannose carbo-

hydrate moieties are attached at residues 917 of the a

chain (Man8GlcNac2πMan9GlcNac2) and 63 of the b chain

(Man5GlcNac2πMan6GlcNac2) (Fig. 1) and together account

for 1.5% of the molecular weight of C3 (9, 10). A complete

disulfide bridge pattern for C3 has been determined (11, 12)

(Fig. 1): the a and b chains are linked by a single disulfide

bridge. Three linkages are found in C3a and a single bridge

is present in the b chain as well as the C3d portion of the a

chain. The N- and C-terminal regions of the a chain are con-

nected with each other via a disulfide linkage. Of interest is

the fact that six linkages are clustered in the 46 kDa C-ter-

minal peptide of the a chain. With the exception of the C3a

(11) and C3d (13) regions of C3, the three-dimensional

structure of C3 is not yet available. Although methylamine-

treated C3 and C3b have been crystallized (14), data could

be collected only at 7.7 Å resolution.

One of the most intriguing features of C3 is its ability to

attach covalently to acceptor molecules on cell surfaces (15)

via ester or amide linkages (16). This property of C3 is de-

rived from the presence of an intramolecular thioester bond

within the C3d region. The thioester bond is formed during

post-translational modification as a result of intramolecular

transacylation between the thiol group of cysteine and the


Fig. 1. Schematic representation of human C3. The molecule consists CR1, CR2, CR3), regulatory proteins (H and P), and binding proteinsof an a- and b-chain structure linked by a disulfide bond. Proteolytic (factors B and conglutinin) are shown. N-linked high-mannosecleavage sites, disulfide linkages and binding sites for receptors (C3aR, carbohydrates are located at residues 63 and 917.

g-amide group of the glutamine within the C3 sequence Gly-

Cys988-Gly-Glu-Gln991-Asn (Fig. 1) (17). Interestingly, the

highly conserved thioester domain is encoded by class 1-1

exons, the type most commonly reshuffled and duplicated

(18).

It is believed that in native C3 the thioester bond is pro-

tected within a hydrophobic pocket and is exposed only in

the C3b fragment upon cleavage of C3 by C3 convertases. This

belief is supported by the fact that the rate of spontaneous

hydrolysis of the thioester bond is very slow under physiol-

ogic conditions and has been estimated to be 0.005%/min

(19). It has been estimated that, at any given time, 0.5% of

the total C3 present in fresh human plasma is present in its

hydrolyzed form (C3H2O) (20). Once C3 is cleaved to C3b,

the transiently exposed thioester bond in C3b (half life ∂100

ms) participates in a transacylation reaction with nucleophilic

37Immunological Reviews 180/2001

groups present on cell surfaces, complex carbohydrates, or

immune complexes (16, 21–24). In many biological systems

the majority of C3b is linked via an ester bond indicating a

strong preference for the hydroxylated targets.

Until recently, attachment of C3b to various acceptors had

been considered a non-specific reaction; however, recent

studies have clearly demonstrated that C3b displays a high

degree of specificity in reacting with targets such as carbo-

hydrates, C3b, C4b, and IgG (21, 22, 25, 26), and that this

specificity can be an important factor in complement acti-

vation (21). Analysis of its reactivity with sugars and alcohols

has clearly revealed that thioesters of C3 show a preference

for the primary OH in the 6 position and the OH group next

to the less bulky group. In addition, in human IgG1 and C4b,

Thr144 and Ser1213 respectively have been identified as the

major, if not the only, sites with which C3b reacts (21, 22,


Fig. 2. Activation and degradation of C3. The cleavage sites areindicated by arrows. The location of N-linked carbohydrate sites areindicated with closed balloons. The molecular weights of thepolypeptides are calculated on the basis of their deduced amino acidsequences (7).

25). These findings demonstrate the selection by C3b of spe-

cific OH groups and specific residues on proteins. Thus, the

belief that metastable C3b reacts randomly or non-specifically

is apparently not correct. Having said this, it should be

pointed out that although C3b shows a preference for certain

hydroxyl groups, it does not have the ability to discriminate

between self and non-self. Thus, in the absence of regulators,

it can bind equally well to host cells, thereby causing damage

to these cells. The attachment of C3b to acceptor molecules


is necessary to initiate the formation of the membrane attack

complex (MAC), phagocytosis of foreign particles, enhance-

ment of humoral responses to antigens, and probably elimin-

ation of self-reactive B cells (27).

Proteolytic activation of native C3 by either the classical/

lectin (C4b,2a) or alternative (C3b,Bb) pathway C3 convertas-

es leads to cleavage between residues 726 and 727 (Arg–Ser)

and generation of C3b (Mr 176,000) and C3a (Mr 9,000)

(Figs 1 & 2). In contrast to native C3, C3b expresses multiple

binding sites for other complement components, including

C5; properdin (P); factors H, B, and I; complement receptor

1 (CR1); and the membrane co-factor protein (MCP) (28).

Binding of these proteins to C3b leads either to amplification

of the C3 convertase (by B and P in the presence of factor D)

and initiation of the MAC (C5b-9) or to the inactivation of

C3b (by factor I in the presence of H, CR1, and MCP) (1).

Whether amplification or inactivation occurs depends on the

nature of the surface to which the C3b is attached.

The downregulation of C3b by factor I proceeds in three

steps (29) and requires one of the several co-factor molecules

(MCP, CR1 or H) (Figs 1 & 2). Cleavage of the aø chain of C3b

occurs in two places: first between residues 1281 and 1282

(Arg–Ser) to generate iC3b1 and then between residues 1298

and 1299 (Arg–Ser) to liberate the C3f fragment (Mr 2,000)

and yield iC3b2 (30–32). A third factor I cleavage site, with

CR1 or factor H serving as the co-factor (30, 31), has been

reported to exist at residues 932–933 (Arg–Glu) of the a

chain of C3; cleavage here generates the C3c and C3dg frag-

ments (32). It was previously thought that the first two cleav-

ages are necessary to inactivate C3b, but recently our labora-

tory has shown that the first cleavage by itself is sufficient to

inactivate C3b (33).

Studies of C3–ligand interactions

C3 is a large molecular weight protein (Mr 185,000) that

interacts with at least 25 different soluble and membrane-

bound proteins. Thus, identification of its binding sites for

various ligands is a challenging task. Our laboratory has

chosen a multiattack approach. 1) The first strategy involved

localization of binding sites on C3 by studying the molecule

at the protein level through degradation of C3 either enzy-

matically or chemically, analysis of protein fragments for

binding to various C3-binding proteins, identification and

characterization of the fragments, of interest and validation of

the data using synthetic peptides and analogs. 2) The second

approach originated at the DNA level and involved construc-

tion of an expression minilibrary (∂200–300 bases) from


the cDNA spanning the entire coding sequence of C3 (34).

The library was screened with anti-C3 antibodies known to

block specific interactions and also with various proteins that

bind C3. The reactive clones were sequenced, allowing the

mapping of specific sites. 3) The third approach involved

studies of the evolution and conservation of binding sites

within the C3 molecule from various species and other

homologous proteins, such as C4, C5, and a2-macroglobulin.

Here, comparison of the C3 amino acid sequences among

species and correlation of this information with the ability of

these C3s to bind various ligands and receptors were used

to design site-directed mutagenesis and chimeric molecule

experiments (29). A similar approach, referred to as the

INDEL approach, was used by Ogata and colleagues to ident-

ify the functionally important regions of C3 and C5 (35, 36).

Using the strategies outlined above, we have successfully

identified the binding sites on C3 for properdin (37), bovine

conglutinin (38), factor H (39), CR1 (40), and CR2 (41).

The plasma glycoprotein properdin stabilizes C3 convertase

and allows rapid amplification of surface-bound C3b. Our ef-

forts to localize the properdin-binding site in C3b were

greatly assisted by a phylogenetic analysis of C3. Initial studies

showed that properdin binds to both C3b and C3c (42), and

subsequent studies have placed the properdin binding site

within residues 1385–1541 of C3 (43). Comparison of the

amino acid sequences of human, mouse, and rabbit C3 (all

of which bind to human properdin) with those of human

and mouse C4, C5, and a2-macroglobulin (homologous but

non-properdin-binding proteins) identified a region (resi-

dues 1402–1435 of the human C3 sequence) that was con-

served and therefore a possible candidate for the properdin-

binding site. We have shown that a synthetic peptide

(C31402–1435) corresponding to this segment of C3 binds to pro-

perdin, inhibits properdin binding to C3, and inhibits the

activation of the alternative pathway by rabbit erythrocytes

(37). Our data reinforce the belief that properdin stabilization

of the C3 convertase is necessary for efficient amplification of

the enzyme cascade during alternative pathway complement

activation and are in agreement with studies of patients with

properdin deficiency, which have indicated that properdin is

essential for optimal complement activation (44).

To date the N-terminal region (residues 727–768) of the

aø chain of C3b is considered to be one of the most important

regions of C3 because it is known to interact with multiple

proteins. Using C3 fragments, synthetic peptides, and anti-

peptide antibody, we and others (45, 46) have shown that

this region encompasses the binding sites for CR1, factor H,

and factor B. To further probe the importance of this region,


in a subsequent study we have constructed chimeric mol-

ecules in which residues 727–768 were substituted with a

corresponding segment of C3 from a species whose C3 either

binds or does not bind to a specific ligand. In addition, we

also engineered and expressed a deletion mutant in which

residues 727–768 were deleted, and then asked: do the co-

factor molecules (CR1, H, and MCP) bind to multiple sites

on C3 in order for factor I to cleave C3b at multiple sites? Do

the co-factor molecules (CR1, H and MCP) share the same

binding sites on C3? Does factor B bind to the N-terminal

region (residues 727–768) of the aø-chain of C3b?

A careful analysis of the data led us to conclude the fol-

lowing. 1) A major binding site for CR1 and factor H is

located in the N-terminal region of the aø chain of C3b, and

both proteins interact with C3b via a second site. This view

is consistent with a recent report demonstrating involvement

of different regions of C3 in factor H binding (47). 2) MCP

protein binding sites in C3b differ from those for CR1 and

H, thus supporting the idea that binding sites on C3b are

different for some co-factors. 3) Although site-directed muta-

genesis studies performed by others (46) have suggested an

involvement of charged residues of the N-terminal region in

factor B binding, other residues must also contribute signifi-

cantly to factor B binding (29).

Recently, Oran & Isenman (48) confirmed our conclusion

that residues 727–768 are not involved in the interaction with

MCP. They also identified the residues of the N-terminal re-

gion that are important for factor H and CR1 interactions.

Their study demonstrated that the factor H interaction in-

volved residues E744 and E747, whereas the CR1 interaction in-

volved two classes of residues: The first class, consisting of

the E736/E737 pair, E747, and the E754/D755 pair, was found to be

important for the first two factor I cleavages (that result in

generation of iC3b2); the second class, consisting of residue

E744 and the pair K757/E758, were important for the third factor

I cleavage (resulting in the generation of C3dg) (48).

In recent years the interaction of complement receptor 2

(CR2) with its ligand C3dg, a proteolytic fragment of C3, has

received considerable attention not only from the scientists in

the complement field but also from the researchers outside

the field who are interested in B-cell biology. CR2 is a 140

kDa glycoprotein expressed on B cells, on some T cells, and

on follicular dendritic cells (49–51). The involvement of

complement in the humoral immune response was first ob-

served more than 2 decades ago (5); however, this report did

not receive much scientific attention. The recent availability

of C3 knockout mice has allowed complementologists to re-

visit this issue. C3 knockout mice have been found to produce


an impaired antibody response to the T-cell-dependent jX

174 antigen, with a response characterized by a reduced

number and size of germinal centers (52). Similarly, mice

with a disrupted CR2 locus showed impaired antibody re-

sponse to jX 174 (53) and sheep red blood cells (54). In

addition, administration of anti-CD21 antibody or soluble

CD21 suppressed the in vivo immune response (55–57).

In general, invading pathogens, antigens, or immune com-

plexes activate complement pathways, leading to covalent

attachment of complement C3 to these pathogens/antigens/

immune complexes (21, 22). Whether the antibody-en-

hancing function of complement is mediated primarily by

attachment of complement C3 to antigen was elegantly inves-

tigated by Fearon and his colleagues (58). They coupled hen

egg lysozyme (HEL) to multiple copies of C3d (a proteolytic

fragment of C3dg) and injected this model antigen into mice

without adding adjuvant. HEL attached to two or three copies

of C3d was 1,000- or 10,000-fold more immunogenic, re-

spectively, than HEL alone (58). Thus, it became clear from

their study that C3d acts as a ‘‘natural adjuvant‘‘

Previously we localized a CR2-binding site to residues

1201–1214 of the C3d region of C3 (41). Using a site-di-

rected mutagenesis approach, Diefenbach & Isenman analyzed

the involvement of eight of the 11 residues of this region

(62). They found that substitution with alanine at positions

1999–1200 (ED), 1203–1204 (KQ) or 1207–1208 (NV) had

no effect on the binding of C3 mutants to CR2, whereas a

triple mutant containing substitutions at all six residues

showed a 20% reduction in binding as compared to that for

wild-type C3. From these data they inferred that this region

is not involved in CR2 binding. However, several other studies

point toward the involvement of this region in CR2 binding.

a) A synthetic peptide corresponding to this region binds to

Raji cells in a CR2-dependent manner (41). b) The CR2-bind-

ing site in Epstein–Barr virus (EBV) gp350/220 (a viral pro-

tein that binds to CR2) is homologous to these residues, and

mutation in this region of the intact EBV gp350/220 mol-

ecule abolishes the binding to CR2 (61). c) In the study by

Diefenbach & Isenman (62), a mutant that is substituted at

six residues (ED, KQ and NV) with alanine displayed a 20%

reduction in binding to CR2. d) It has been demonstrated

that C3d peptide K1195-A1210 when coupled to anti-idiotype

antibody, induces a strong idiotype and antigen-specific re-

sponse (59).

Using human/trout as well as trout/human C3dg chim-

eras, we recently found that both the N- and C-terminal

halves of human C3dg are important for CR2 binding (63),

and that a number of weak interactions may have a cumulative


effect on C3dg binding to CR2. Very recently, Clemenza et

al. (64) studied the role of charged residues in C3dg in the

interaction with CR2. Their data suggested that mutation at

charged residues E1008/E1010 pair, E1131, D1134 and E1137, and I1135

results in a significant loss in CR2 binding. It should be noted

that the human/trout chimera, which contains all these resi-

dues, binds only at low ionic strength, suggesting that resi-

dues other than these six must be involved. We believe that

detailed structural design of C3d mutants based on the crystal

structure and species specificity data, together with analysis

of the X-ray structure of informative mutants, as well as the

development of technologies that will allow measurement of

the dynamics of protein–protein interactions should give us

a global picture of the C3d–CR2 interaction.

C3 origin, evolution, and diversity

A thorough understanding of any biological system requires

detailed knowledge of its origin, evolution, and diversity.

Since C3 is essential for activation of all three complement

pathways, it is not unreasonable to expect that understanding

its origin would shed light on the origin of the complement

system.

At present, a prevailing hypothesis is that complement pro-

teins C3, C4, and C5 were generated by gene duplication from

a2-macroglobulin. According to this hypothesis, one of the

duplicated a2-macroglobulin genes was modified to form a

C3/C4/C5 ancestral gene, which was then duplicated a second

time to form the C4 and C3/C5 genes. Finally, the C3/C5 gene

was duplicated to produce the C3 and C5 genes. This hypothesis

is based on the sequence similarity and exon–intron organiza-

tion of these proteins (65–67). It is, however, not known at

what point in evolution the first gene duplication took place.

Thus, probing the molecular structure and functions of these

proteins at various stages of the phylogenetic tree should lead

to a detailed understanding of the origin of these proteins in

particular and of the complement system in general.

To study the evolution of C3 and to probe the origin of C3

we characterized C3 from a number of animal species. Re-

search in this direction suggested that in lower vertebrates,

complement may compensate for primitive adaptive response

by providing greater diversity (68). Over the course of 15

years, we have purified C3 molecules from pig, rabbit,

mouse, chicken, cobra, Xenopus, axolotl, sea bream, trout, and

sea urchin and have studied their biochemical and functional

properties. In addition, we have cloned the genes encoding

chicken (69) and Xenopus C3 (70) as well as three trout (Salmo

gairdneri) isoforms of C3 (C3-1, C3-3 and C3-4) (71).


During the process of cloning the gene encoding trout C3

(now named C3-1), we obtained a PCR product whose de-

duced amino acid sequence was only 50% similar to the cor-

responding sequence of trout C3-1. These results prompted

us to hypothesize that trout contain multiple forms of

C3s/C3-related proteins. This finding was particularly note-

worthy because all the functionally active C3 molecules that

had been isolated from various species until that time were

thought to be the products of single genes. Our effort to

isolate these molecules revealed the presence of two new

functionally active isoforms of C3 (C3-3 and C3-4) in trout

(72). Efforts in other laboratories led to the identification of

an additional isoform, C3-2, which was functionally inactive

and showed a tryptic map that differed by 20% from that of

C3-1 (73).

In the carp (Cyprinus carpio) at the DNA level, as many as

eight different polymerase chain reaction clones showing

85–90% amino acid identity with each other and homology

to C3 from other species have been characterized (74). Al-

though it is not certain at this point whether these are allelic

variants or products of different genes, all the clones were

derived from a cDNA library derived from a single fish. Trout

and carp are tetraploid fish; therefore, it appeared that the

presence of multiple forms of C3 might be unique to the

tetraploid condition. However, multiple forms of C3 were

also found in the gilthead sea bream (Sparus aurata) and medaka

fish (Oryzias latipes), both of which are diploid teleost fish,

suggesting that this phenomenon is not unique to the tetra-

ploid fish. This finding also suggests that genes encoding for

these isoforms must have been fixed into the genome of these

animals. We would like to point out here that the presence of

multiple forms of proteins in these fish is not restricted to

C3, since two forms of factor B/C2 molecules have been iden-

tified in trout (75). It would be interesting to study the mech-

anism through which these multiple forms of complement

proteins have been generated.

Since C3 is present in multiple forms in fish, the obvious

question is: do these C3s differ functionally? Analysis of the

binding of these C3 isoforms to various complement-activat-

ing surfaces has shown that these C3s bind with different

efficiencies to various surfaces. For example, in the trout, all

three C3 isoforms bind to various erythrocyte surfaces and to

E. coli to varying degrees, but only C3-1 binds to zymosan.

Similarly, in the gilthead sea bream, C3-1 and C3-2 bind to

zymosan, whereas C3-3, C3-4, and C3-5 fail to bind (76).

These results suggest that the specificity of the thioester bond

in these C3s varies considerably. Such a precedent has already

been established in the case of human C4 isotypes: human


C4A shows strong reactivity with amino groups, whereas C4B

displays a preference for hydroxyl groups (77, 78). Analysis

of the binding mechanism at the molecular level has indicated

that C4B uses a two-step mechanism.Upon C4B activation,

H1106 attacks the internal thioester to form an acyl–imidazole

bond. The released thiol then acts as a base to catalyze the

transfer of the acyl group from the imidazole to the hydroxyl

of the acceptor molecule. Such reactions are also known to

occur in the case of C3 (79).

Alignment of trout C3 isoforms with various C3s and hu-

man C4B has shown that the His1126 residue (equivalent to

H1106 of human C4B) is conserved in C3-1 and C3-3 but is

substituted by Thr in C3-4. Thus, it is possible that the pres-

ence of the His residue in C3-1 and C3-3 would generate a

preference for hydroxyl groups. Recently it has also been

shown that the Glu residue located two amino acids down-

stream from the His1126 forms a hydrogen bond with H1126,

which renders H1126 a stronger nucleophile. This situation may

increase the rate of acyl–imidazole formation in C3 and pro-

mote its specificity for hydroxyl groups, in comparison to

C4B (H1126, Ser1128) (13). Alignment of trout C3s with human

C3 has shown that although this residue is conserved in trout

C3-1, it is substituted by Thr and Ser in C3-3 and C3-4,

rendering C3-1 more like human C3. Whether these differ-

ences in the isoforms indeed correlate with differences in

thioester reactivity among the various trout C3 molecules re-

quires further investigation.

It is now clear that both tetraploid and diploid fish contain

multiple forms of C3, a situation which has led us to ask why

fish demonstrate such structural and functional C3 diversity.

As previously mentioned, structural diversity in C3 allows

these molecules to recognize a broader range of activating

surfaces. Thus, it is reasonable to presume that this diversity

would allow C3 to react with a wider repertoire of micro-

organisms and would therefore enhance fish’s defense against

pathogens (68). This diversity could also play a vital role in

the survival of the animals, because in fish the antibody re-

sponse is represented only by IgM antibodies. Moreover, these

antibodies are of low affinity and limited heterogeneity, and

their response is impaired during the winter. In contrast, the

complement system in these animals can be activated in tem-

peratures as low as 4æC (80).

Virus–C3 interactions

Unlike other pathogens, viruses have to depend on their hosts

for their replication. Thus, in order to survive and multiply

they must overcome the formidable defense mechanisms of


their hosts, including the complement system, which is one

of the major defense mechanism against viruses. The comple-

ment system serves as both an innate and an acquired im-

mune defense against viral infection. Activation of comple-

ment in the presence or absence of antibodies can lead to

virus neutralization, phagocytosis of C3b-coated viral par-

ticles, lysis of infected cells, and generation of inflammatory

and specific immune responses. During the co-evolution of

the viruses with their hosts over several million years, viruses

have not only developed mechanisms to control the comple-

ment system but have also turned these interactions to their

own advantage. Since C3 is a common denominator in the

activation of all three pathways of the complement system, it

became the obvious target for viruses (81). Important

examples of viruses that have developed mechanisms against

C3 include vaccinia, herpes simplex virus (HSV) type 1, and

type 2 (HSV-2).

Vaccinia virus encodes a protein that is homologous to

human complement control proteins. This protein, named

the vaccinia virus complement control protein (VCP) (82),

is one of the two major proteins secreted by vaccinia virus-

infected cells. VCP received scientific attention when it was

discovered that an attenuated mutant of vaccinia virus does

not secrete this protein. Sequence analysis revealed that it

is structurally related to members of the RCA family (82).

VCP, which is encoded by the C3L open reading frame

(ORF) of the vaccinia genome, is a 27 kDa protein that is

composed entirely of four tandemly repeating domains

called short consensus repeats (SCRs) or complement con-

trol protein repeats (CCPs) (83). VCP apparently protects

the infected cells and released virions from attack by host

complement. Evidence for this theory has come from ex-

periments in which VCP was shown to abrogate comple-

ment-mediated antibody-dependent neutralization of vac-

cinia virions (84). In addition, studies using recombinant

vaccinia viruses that do not express VCP have clearly shown

that these viruses are attenuated in vivo (84). A culture me-

dium containing secreted VCP has been shown to inhibit

complement-mediated lysis of sheep erythrocytes, to bind

to C3b and C4b, and to accelerate the decay of the classical

as well as the alternative pathway C3 convertases (83, 85).

To understand the detailed mechanisms by which VCP in-

activates complement, our laboratory has generated a recom-

binant form of VCP (33). A comparison of its effect on com-

plement to that of human factor H and sCR1 has revealed that

the recombinant VCP is less effective than CR1 in inhibiting

the classical and alternative pathways of complement and less

effective than factor H in inhibiting the alternative pathway.


However, it is noteworthy that on a molar basis this VCP was

four times more effective than factor H in inhibiting the

classical pathway, possibly because of its dual action on C3

and C4. Our study also demonstrated that VCP supports the

factor I-mediated cleavages of C3b and C4b. It is known that

CR1 and factor H support the factor I-mediated cleavage of

C3b between Arg1281and Ser1282 (site 1), Arg1298and Ser1299 (site

2), and Arg932and Glu933 (site 3) (Figs 1 & 2) (1). Analysis of

VCP-supported cleavages of C3b showed that it primarily

serves as a co-factor for the first site, leading to the generation

of C3b through a single cleavage (iC3b1) (33). The factor I

co-factor activity of VCP for C4b was similar to that of CR1.

Purification and functional analysis of the VCP-generated

iC3b1 showed that it was unable to interact with factor B to

form the alternative pathway C3 convertase (C3b,Bb) (33).

These results demonstrate that the interaction of VCP with

C3 is different from that of all the other factor I co-factors

characterized to date.

Recent studies have shown that extracellular vaccinia virus

incorporates host complement control proteins into its envel-

ope (86), suggesting that apart from VCP it possesses ad-

ditional mechanisms to evade complement attack. Like VCP,

proteins that have four SCRs and are also homologous to com-

plement control proteins have been found in various mem-

bers of the poxvirus family, including smallpox virus (87,

88), herpesvirus saimiri (HVS) (89, 90), Kaposi’s sarcoma-

associated herpesvirus (HHV-8) (91), and murine g-her-

pesvirus 68 (92). Currently, our laboratory is attempting to

decipher the role of complement control protein homologs

of HVS and HHV-8 in immune evasion.

Viruses have also developed proteins that are structurally

different from CCP but also function to inactivate comple-

ment. Examples include glycoprotein C (gC) molecules of

HSV-1 and HSV-2, which are expressed on the virion envel-

ope and on the surface of infected cells (93–96). Glyco-

protein C of HSV-1 (gC-1) and HSV-2 (gC-2) are highly gly-

cosylated proteins with numerous O-linked oligosaccharides

and nine and seven potential sites for N-linked glycosylation,

respectively (97). Both proteins bind to C3b when they are

expressed on the surface of transfected cells or as purified

proteins (98). It is interesting to note that unlike other regu-

lators of complement activation (factor H, MCP, decay acceler-

ation factor (DAF) and CR1), which interact with C3b, both

gC-1 and gC-2 bind to native C3 (99).

Studies on the mechanism of gC interaction with comple-

ment have shown that gC-1, like other regulators of comple-

ment activation (factor H, CR1, and DAF), accelerates the decay

of a bimolecular C3 convertase (C3b,Bb) into its subunits;


however, in contrast to factor H and CR1, it does not mediate

the proteolytic inactivation of C3b by factor I (100). It has also

been shown that gC-1, but not gC-2, can inhibit the binding of

C5 to C3b and destabilize C3 convertase by inhibiting the bind-

ing of properdin to C3b (101). Structure–function analysis of

the gC-1 molecule has shown that the C5 and P-blocking do-

main is located near the amino terminus, while the C3-binding

domain is found in the central region of the molecule (99,

101). Recent in vivo studies on the role of complement-inter-

acting domains of gC-1 has clearly shown that the C3 binding

domain is more important than the C5/P-blocking domain and

is a major contributor to complement evasion (102).

In addition to the above-mentioned mechanisms, some vi-

ruses have also developed ways to mimic C3 and interact with

complement receptors to aid their entry into cells (103). One

of the important and well-studied examples of this group is the

EBV, which infects B cells and epithelial cells through CR2

(104). The protein on EBV that is responsible for binding to

CR2 is gp350/220; both C3d (a natural ligand of CR2) and

gp350/220 interact with first two SCRs of CR2 (105). As de-

scribed before, one of the binding sites in C3d is located be-

tween residues 1201 and 1214 (PGKQLYNVEATSYA) (41). A

sequence similar to this C3d sequence has also been identified

in gp350/220, suggesting that the C3d and EBV binding sites

on CR2 are either identical or conformationally related. A series

of studies using monoclonal antibodies, purified proteins, and

site-directed mutagenesis of the proposed sequence has con-

firmed that the binding site for EBV on CR2 is located within

the C3dg-like sequence of gp350/220 (61). Other viruses that

use complement receptors for cellular entry include measles,

echo, and West Nile viruses. These viruses use membrane co-

factor protein, DAF, and complement receptor type 3, respec-

tively, to initiate infection (103, 106, 107).

C3-based inhibitors

Complement proteins that are involved in the activation pro-

cess, including C3, do not have the ability to discriminate

between the self and the non-self. Thus, they have the poten-

tial to destroy any cells to which they bind, including those

of the host. As a means of preventing this destructive activity,

the complement system is tightly controlled by regulatory

proteins such as C1 inhibitor, factor H, C4bp, CR1, DAF, MCP,

carboxypeptidase N, and CD59. Therefore, it is not surprising

that unregulated activation of the complement system leads

to host cell damage (108, 109).

At present many basic research scientists and applied scien-

tists in the biotechnology field and larger pharmaceutical


firms are interested in developing complement inhibitors that

would block various steps of its activation. We have focused

our attention on C3, since inactivation of this protein would

lead to inhibition of C3a and C5a generation and of C5b-9

formation, all of which are implicated in complement-me-

diated damage to host cell (110). Although others are devel-

oping high molecular weight complement proteins (CR1,

DAF, and MCP) to block complement-mediated damage, we

are interested in developing small molecular weight inhibitors

because they are cost-effective, have better tissue penetration,

and can be developed for oral use.

In our initial efforts we used combinatorial peptide librar-

ies to identify C3-interactive peptides, with the goal of identi-

fying C3-binding peptides that would functionally mimic

other C3-regulating proteins. Our approach led to the identi-

fication of a novel 13-residue cyclic peptide (111), later

named compstatin. Unlike natural inhibitors of complement

that act on C3b, compstatin binds to native C3 and inhibits

its cleavage by C3 convertase (111). Most importantly, this

inhibition is not caused by sterically hindered access to the

C3a/C3b cleavage site (111). Recently, an independent study

has confirmed our results (112). We have observed that the

kinetics of compstatin binding to native C3 do not follow a

1:1 Langmuir binding model; instead, the binding data fits

well to a two-state conformational change model. Whether

such a binding mechanism is important for compstatin’s ac-

tivity needs further investigation (113).

Thus far, compstatin has been tested in three different clin-

ically relevant models. 1) Hyperacute rejection in discordant

kidney xenotransplantation has been studied ex vivo in a por-

cine-to-human perfusion model. In this model, compstatin

significantly prolonged the survival of the kidneys (114,

115). 2) The effect of compstatin has also been tested in

models of extracorporeal circulation (116), where it effec-

tively inhibited the generation of C3a and sC5b-9 and the

binding of C3/C3 fragments to a polymer surface. As a result

of the inhibition of complement activation, the activation of

polymorphonuclear leukocytes (as assessed by the expression

of CD11b) and the binding of these cells (CD16π) to the

polymer surface were almost completely lost (116). 3) Most

recently, compstatin has been tested in vivo in primates to

examine its effect on complement activation induced by a

heparin-protamine complex; here it effectively inhibited

complement activation (117).

Structure-based rational design of peptidomimetics and

crafting of small molecule inhibitors requires knowledge of

the complete 3D of the peptide inhibitor and the target pro-

tein. We have achieved the first step towards this goal by de-


termining the 3D structure of a major conformer of compsta-

tin in solution by two-dimensional NMR (118). Although the

peptide in its current form is effective in vivo, the structural

information obtained for compstatin is being used for the

rational design of a small-molecule inhibitor that can be ad-

ministered orally.

Concluding remarks

As described above, a wealth of information has been ob-

tained regarding the structure of C3 and its interactions with

ligands. Nevertheless, the information obtained is essentially

one-dimensional, in part because C3 is a large molecular

weight protein and, with the exception of C3a and the C3d

region of C3 its three-dimensional (3D) structure is still not

available. The studies conducted thus far have utilized a var-

iety of approaches to define the binding sites on C3, includ-

ing generation of synthetic peptides corresponding to various

regions of C3, development of monoclonal and site-specific

antipeptide antibodies against C3, site-directed mutagenesis,

and expression of chimeric molecules. Each of these methods

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2. 7. 12.Al-Sharif WZ, Sunyer JO, Lambris JD, Smith De Bruijn MHL, Fey GH. Human Dolmer K, Sottrup-Jensen L. DisulfideLC. Sea urchin coelomocytes specifically complement component C3: cDNA coding bridges in human complement componentexpress a homologue of the complement sequence and derived primary structure. C3b.component C3. Proc Natl Acad Sci USA FEBS Lett 1993;315:85–90.J Immunol 1997;160:2983–2997. 1985;82:708–712. 13. Nagar B, Jones RG, Diefenbach RJ, Isenman

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has its limitations. Therefore, conclusions concerning the in-

volvement of a particular region in ligand binding can be

drawn only if the various methods point in a single direction.

We believe that the identification of the functional domains

of C3 by the methods discussed above, together with a deter-

mination of their 3D structure and the development of tech-

nologies that will allow determination of the dynamics of

protein–protein interactions are needed if we are to gain a

complete understanding of C3–ligand interactions. Although

this approach is ambitious, it is the only way to obtain 3D

structural information regarding a large, architecturally com-

plex and multifunctional protein such as C3.

Because C3 emerged millions of year ago, it is reasonable

to hypothesize that it may participate in ‘‘non-immune’’

mechanisms. For example, it has recently been shown to be

expressed in the regenerating limb blastema cells of urodeles

(119), suggesting that it plays a role in regenerative processes.

In an another study, C5 has been implicated in liver regenera-

tion (120). Further studies in this direction may shed light

on ‘‘new’’ functions of C3 in particular and of the comple-

ment system in general.


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Structure and biology of complement protein C3, a connecting link between innate and acquired immunity